All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The hybridization of single-stranded DNA and RNA to complementary immobilized probe strands is being used for detection of known sequences associated with pathogens and mutations. The immobilization of biological polymers on solid supports has also had significant impact on drug discovery and medical diagnostic methods. By using an array of different immobilized probe DNA strands, one may search for many types of DNA or RNA in parallel or determine the entire sequence of a single long strand of DNA or RNA [1-5]. U.S. Pat. No. 5,143,854 [15] describes the large scale photolithographic solid phase synthesis of polypeptides. The polypeptide arrays are synthesized on a substrate by attaching photo-removable groups to the surface of a substrate, exposing selected regions of the substrate to light to activate those regions, and attaching an amino acid monomer with a photo-removable group to the attached regions. The steps of activation and attachment can be repeated until polypeptides of desired length and sequences are synthesized. U.S. Pat. No. 5,412,087 [8] describes substrates with surfaces comprising compounds with thiol functional groups at one end, and protected at the other end with a photo-removable protecting group which can be used to construct arrays of immobilized anti-ligands, such as oligonucleotide probes or other biological polymers. A commercially available product (offered by Affymetrix, 3380 Central Expressway, Santa Clara, Calif. 95051) is made by the light-directed synthesis of DNA [6-9]. According to the current state of the art pertaining to the synthesis of polynucleotide arrays, there is only a 92-94% chance that a new nucleotide will be incorporated where desired [16]. Current technology thus imposes certain constraints on the possible array configuration, such as the practical upper limit on the number of nucleotides being approximately ten.
Presented here is a novel method for making DNA molecules of desired nucleotide sequence, and an alternate, more efficient method for the synthesis of DNA arrays which utilizes local melting of hybridized DNA rather than photochemistry to achieve a known pattern of DNA strands. Using the methods of this invention, one skilled in the art will be able to make DNA hybridization probes with longer nucleotide strands and with greater fidelity than is now available using current technology. One possible advantage of longer strands would be the stable hybridization of target strands to the hybridization array at room temperature. This would permit the use of annealing cycles for the purpose of improving the fidelity of the hybridization.